Plant-derived elastin binding protein ligands and methods of using the same

ABSTRACT

The present invention describes novel plant derived elastin-like peptides and peptidomimetics that may serve as functional ligands for elastin receptors and stimulate elastogenesis. The novel plant derived peptides provide an alternative (non-animal derived) source of GXXPG (SEQ ID NO. 2) containing peptides. The present invention also describes therapeutic compositions containing novel plant derived peptides or peptidomimetics useful in stimulating elastogenensis and capillary dilatation. The therapeutic compositions of the present invention that comprise novel plant derived peptides or peptidomimetics may be combined with other therapeutic agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of pending U.S.application Ser. No. 12/781,710 filed May 17, 2010, which is acontinuation of U.S. application Ser. No. 11/405,843, filed Apr. 17,2006, now issued as U.S. Pat. No. 7,723,308, on May 25, 2010, whichclaims the benefit of U.S. Provisional Application No. 60/671,557, filedApr. 15, 2005, and U.S. Provisional Application No. 60/737,586, filedNov. 17, 2005, all of which are hereby incorporated by reference intheir entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

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JOINT RESEARCH AGREEMENT

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INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A CD

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BACKGROUND OF THE INVENTION

Elastin is an amorphous protein present in the elastic fibers of tissuessuch as arteries, blood vessels, skin, tendons and elastic ligaments,the abdominal wall, and lungs. Unlike other fibrous tissues likecollagen, elastin is unique in that it may be stretched to over 150percent of its original length but it can rapidly return to its originalsize and shape. This property of elastin provides tissues thatincorporate it the ability to resume their original form afterstretching due to, for example, blood flow, breathing, or bending. Likecollagen protein, elastin contains about 30% glycine amino acid residuesand is rich in proline. Elastin differs from collagen in that itcontains very little hydroxyproline or hydroxylysine. Elastin has a veryhigh content of alanine and also contains two unique amino acidsisodesmosine and desmosine. These amino acids are believed to beresponsible for elastin's ability to return to its original shape afterstretching.

A lack of elastin or genetic abnormalities affecting elastic fibers inskin, as evidenced in Costello Syndrome, Cutis Laxa and PseudoxanthomaElasticum, for example, lead to premature aging most noticeablycharacterized by wrinkling and folding of the skin in children(pre-teenage) suffering from these illnesses. Given that theseconditions only affect elastic fibers in skin, it is highly probablethat development of wrinkles in aged skin is due to damage to or loss ofelastic fibers in skin. Unfortunately, dermal fibroblasts lose theirability to make elastin (the major component of elastic fibers) by theend of puberty. Hence, adult dermal fibroblasts cannot repair or replacedamaged elastic fibers in skin later in life, leading to an essentiallyirreversible formation of wrinkles

The protein motif VGVAPG (SEQ ID NO. 1) has been previously shown tostimulate proliferation/migration of monocytes, dermal fibroblasts, andsmooth muscle cells through its interaction with the cell-surfaceelastin receptor. Other GXXPG (SEQ ID NO. 2) sequences recognized by BA4antibody are also known ligands for the elastin receptor. More recently,it has been shown that elastin peptides, liberated through proteolyticdigestion of bovine ligamentum nuchae and containing elastin receptorligand sequences (GXXPG) (SEQ ID NO. 2) also induce elastogenesis indermal fibroblasts through interaction with the elastin receptor.

SUMMARY OF THE INVENTION

Given the concern associated with bovine derived tissue, an alternativesource of GXXPG (SEQ ID NO. 2) containing peptides that might alsoinduce elastogenesis in dermal fibroblast cells would be useful.Embodiments of the present invention relate to a digest of plant-derivedelastin-like protein and synthetic peptides mimicking these sequencesdetected in plants which interact with elastin receptors and appear tostimulate elastogenesis. In some embodiments, the plant-derivedelastin-like protein is derived from rice bran.

The invention also comprises peptides that enhance deposition of elastinor appear to stimulate elastogenesis. In one embodiment, a peptide ofthe invention stimulates elastogenesis. In one embodiment, the peptideis a synthetic sextapeptide. In another embodiment, the peptide is asynthetic linked sextapeptide.

In preferred embodiments, the sextapeptide comprises the sequenceX₁-X₂-X₃-X₄-X₅-X₆ (SEQ ID NO. 3), wherein X₁ is V or I, X₂ is G, X₃ is Aor L, X₄ is M or S, X₅ is P and X₆ is G. In further embodiments, alinked sextapeptide is provided comprising one or more linking aminoacid residues wherein the linking residues join two sextapeptidecompounds, each sextapeptide having the sequence X₁-X₂-X₃-X₄-X₅-X₆ (SEQID NO. 3), wherein X₁ is V or I, X₂ is G, X₃ is A or L, X₄ is M or S, X₅is P and X₆ is G. In one embodiment, the sextapeptide of the inventioncomprises VGAMPG (SEQ ID NO. 4), VGLSPG (SEQ ID NO. 5), IGAMPG (SEQ IDNO. 6), or IGLSPG (SEQ ID NO. 7). In one embodiment, a linkedsextapeptide (or sextapeptide dimer) of the invention comprisesVGAMPGAAAAAVGAMPG (SEQ ID NO. 8), VGLSPGAAAAAVGLSPG (SEQ ID NO. 9),VGVAPGAAAAAVGVAPG (SEQ ID NO. 10), IGAMPGAAAAAIGAMPG (SEQ ID NO. 11), orIGLSPGAAAAAIGLSPG (SEQ ID NO. 12).

In another embodiment, a sextapeptide is provided that comprises thesequence IGVAPG (SEQ ID NO. 13). In one embodiment, a linkedsextapeptide is provided that comprises the sequence IGVAPGAAAAAIGVAPG(SEQ ID NO. 14). Another linked sextapeptide of the invention comprisesthe sequence of two sextapeptides having the sequence IGVAPG (SEQ ID NO.13) that are joined by a linker.

In a further embodiment of the invention the linking moiety can be anymoiety recognized by those skilled in the art as suitable for joiningthe sextapeptides so long as the sextapeptide compound(s) retain theability to interact with the elastin receptor and induce elastogenesis.The linking moiety may be comprised of, for example, but not limited to,at least one of alanine or any other amino acid, a disulfide bond, acarbonyl moiety, a hydrocarbon moiety optionally substituted at one ormore available carbon atoms with a lower alkyl substituent. Optimally,the linking moiety is a lysine residue or lysine amide, i.e., a lysineresidue wherein the carboxyl group has been converted to an amide moiety—CONH.

The present invention also provides non-peptide or partial peptidemimetics of any of the aforementioned synthetic peptides.

Also provided are compositions comprising chemically digested plantpreparations or extracts. In one embodiment, the chemically digestedplant extracts comprise an elastin-like peptide that contain desmosines,crosslinking amino acid characteristic for elastin and never reported inany plant-derived protein. In some embodiments, the chemically digestedplant extracts are chemically digested rice bran extracts. In anotherembodiment, the chemically digested plant extracts induce or stimulateelastogenesis. In yet another embodiment, the invention comprisespreparations comprising a plant-derived elastin-like protein, includingan elastin-like protein from rice bran.

The invention further provides pharmaceutical compositions comprisingthe sextapeptides, linked sextapeptides, peptide mimetics thereof, andchemically digested plant extracts of the invention. The pharmaceuticalcompositions are provided in a therapeutically effective amount. Thetherapeutically effective amount is, in some embodiments, an amount thatstimulates elastogenesis. In further embodiments, the therapeuticallyeffective amount is an amount that stimulates proliferation andmigration of dermal fibroblasts into an area of skin or tissue. Thetherapeutically effective amount may be, according to other embodiments,an amount sufficient to provide an appearance of increased elastogenesisin a tissue.

Also provided are methods of using the compositions of the invention.According to one embodiment, the compositions of the invention can beused to provide an appearance of increased elastogenesis in a tissue.According to another embodiment, the compositions of the invention canbe used to stimulate elastogenesis.

The invention comprises methods of stimulating elastogenesis comprisingadministering a therapeutically effective amount of a composition of theinvention. The composition of the invention can be used to improve theappearance of skin, for example, for removal of facial lines andwrinkles, as well as stretch marks of aged skin. According to oneembodiment, the invention comprises a method of improving appearance ofskin comprising applying a pharmaceutical composition of the inventionto skin in an amount sufficient to stimulate proliferation and migrationof dermal fibroblasts and elastogenesis whereby enhanced elastindeposition in said skin provides elasticity and tone to the skin.Moreover, the compositions of the invention may tighten loose, saggingskin on the face and other parts of the body including arms, legs, chestand neck areas, or give the appearance of reducing wrinkles Othermethods of use of the compounds of the present invention includestimulation of smooth muscle cells and gingival fibroblasts to produceelastin and fibrillin (oxytalan fibers), respectively, for the treatmentof neointimal thickening and loosening of teeth (gingivitis),respectively.

Furthermore, the compositions may be used to enhance wound healing andto prevent and treat cutaneous hypertrophic scars. Accordingly, anotherembodiment of the invention includes a method of promoting wound healingand reducing scarring comprising applying a pharmaceutical compositionof the invention to the wound in an amount sufficient to stimulatedeposition of elastin at a site of injury wherein the elastin holdsinjured tissue together and reduces scarring by providing elasticity andtone to the tissue.

The compositions of the present invention may have either cosmetic ortherapeutic purposes. For example, the compositions of the presentinvention may be used according to another embodiment to treat postinfarct scar. According to this embodiment, the invention includes amethod of treating post infarct scar comprising applying apharmaceutical composition of the invention to a post infarct scar in anamount sufficient to stimulate deposition of elastin at the post infarctscar.

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of necessary fee.

DESCRIPTION OF DRAWINGS

FIG. 1: Primary cultures of human dermal fibroblasts, derived from a 44year-old female, maintained for 7 days in the presence or absence ofsoluble or insoluble rice bran (<3,000 daltons). Cells were fixed andimmunostained with anti-tropoelastin antibodies andfluorescein-conjugated secondary antibodies (GAR-FITC). Panel A:Control. Panel B: Soluble fraction rice extract. Panel C: Insolublefraction rice extract.

FIG. 2: Primary cultures of human dermal fibroblasts, derived from 38and 44 year-old females, maintained for 7 days in the presence orabsence of VGVAPG (SEQ ID NO. 1) (5 μg/mL), EBPL-1 (5 μg/mL), EBPL-2 (5μg/mL) and EBPL-3 (5 μg/mL). Cells were fixed and immunostained withanti-tropoelastin antibodies and fluorescein-conjugated secondaryantibodies (GAR-FITC). Two representative micrographs are shown for eachcondition.

FIG. 3. Quantitative assessment of [³H]-valine incorporation into newlydeposited insoluble elastin in dermal fibroblast cultures maintained for6 days with (SEQ ID NO. 2) GXXPG-containing peptides alone and incombination with copper, manganese and iron salts.

FIG. 4. Elastin deposition in 7 day old dermal fibroblast cultures inthe presence and absence of chemically digested rice bran (CDRB). Tworepresentative micrographs are shown for each condition.

FIG. 5. Results of RT-PCR using specific tropoelastin probe indicatethat EBPL-2 and VGVAPG (SEQ ID NO. 1) stimulated transcription of theelastin gene in adult dermal fibroblasts maintained in serum-free mediumand media supplemented with 10% fetal bovine serum (FBS).

FIG. 6. (A) Results of Morphometric analysis of 7 day old culturesimmunostained with anti-elastin antibody indicate that EBPL-2 and VGVAPG(SEQ ID NO. 1) treated adult dermal fibroblasts maximally produced 710%and 457% more elastic fibers, respectively, than control cultures asassessed by quantitative morphometric analysis of immuno-detectabletropoelastin. EBPL-2-treated fibroblasts maximally produced 141% moretropoelastin than maximum ProK-60-induced levels. (B) Representativemicrographs depicting immuno-detectable elastic fibers (green) incultures of normal dermal fibroblasts (derived from 55 old male),maintained for 7 days in the presence and absence of ProK-60-(25 μg/mL),EBPL-2-(25 μg/mL) or VGVAPG (SEQ ID NO. 1) (10 μg/mL).

FIG. 7. Results of three independent experiments in which confluentcultures of human fibroblasts derived from normal human skin weremetabolically labeled with [³H]-Valine for 3 days and then assessed forthe content of the newly deposited (radioactive) NaOH-insoluble elastin.Statistical analysis indicated that EBPL-2 (10 μg/mL)- andacetylated-EBPL-2 (Ace-EBPL-2, 10 μg/mL)-stimulated adult dermalfibroblasts deposited 36% and 51% more radioactively labeled insolubleelastin, respectively, as compared to non-treated fibroblasts. AmidatedEBPL-2 did not enhance deposition of insoluble elastin.

FIG. 8. Results of three independent experiments in which confluentcultures of human fibroblasts derived from normal human skin weremetabolically labeled with [³H]-Valine for 3 days and then assessed forthe content of the newly deposited (radioactive) NaOH-insoluble elastin.Statistical analysis indicates that both VGVAPG (SEQ ID NO. 1) andEBPL-2 enhanced net deposition of insoluble elastin in a dose dependentmanner and that the effect of the EBPL-2 (maximum 175% over controlvalues) significantly exceeded the effect induced by VGVAPG (SEQ IDNO. 1) (maximum 109% over control values).

FIG. 9. Representative micrographs depicting immunostained (green)elastic fibers deposited in 7 day old cultures of normal skin humanfibroblasts. The rice bran extract sequentially digested with oxalicacid (D) induced more production of tropoelastin than whole insoluble(B) and soluble (C) rice bran in 7 day-old cultures of adult dermalfibroblasts as compared to control cultures (A).

FIG. 10. Both Amylase-digested soluble and insoluble rice branpreparations contain proteins that reacted with antibody raised to humantropoelastin. Additionally, the soluble bran preparation also contains apeptide with the unique AKAAAKAAAKA (SEQ ID NO. 15) domain detected intropoelastin.

FIG. 11. Western blotting with anti-tropoelastin antibody demonstratingthat CNBr digests of rice bran preparations contain protein(s) withsimilar immunoreactivity as human 70-kDa tropoelastin and itsdegradation products. 1: Cyanogen bromide digest of soluble rice bran.2: Cyanogen bromide digest of insoluble rice bran. 3: Extract fromculture of elastin-producing human fibroblasts.

FIG. 12. Representative micrographs of 10-day old organ cultures ofhuman skin explants (44 year woman) stained with Movat pentachromestain. Cultures treated with EBPL-2 contained more elastic fibers (blackstain) produced by cells migrating out of the basal layer of theepidermis, which contain multi-potential stem cells (Upper panel) and bymyofibroblasts localized in the deeper dermal zone (Lower panel).

FIG. 13. Multiple micrographs demonstrating that treatment with EBPL-2induced dilatation of the existing dermal capillaries, and stimulatedproduction of new elastic fibers by pericytes, cells located in theperipheral layer of those capillaries.

FIG. 14. Representative micrographs of 7 day old cultures of human heartstromal cells. The micrographs of FIG. 14 demonstrate that syntheticEBPL peptides significantly increased production of elastic fibers bystromal cells isolated from human heart.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularmolecules, compositions, methodologies or protocols described, as thesemay vary. It is also to be understood that the terminology used in thedescription is for the purpose of describing the particular versions orembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toa “cell” is a reference to one or more cells and equivalents thereofknown to those skilled in the art, and so forth. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. All publications mentioned herein are incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

As used herein, the term “about” means plus or minus 10% of thenumerical value of the number with which it is being used. Therefore,“about 50%” means in the range of 45%-55%.

The term “cosmetic,” as used herein, refers to a beautifying substanceor preparation which preserves, restores, bestows, simulates, orenhances the appearance of bodily beauty or appears to enhance thebeauty or youthfulness, specifically as it relates to the appearance oftissue or skin.

The term “improves” is used to convey that the present invention changeseither the appearance, form, characteristics and/or the physicalattributes of the tissue to which it is being provided, applied oradministered. The change in form may be demonstrated by any of thefollowing alone or in combination: enhanced appearance of the skin;increased softness of the skin; increased turgor of the skin; increasedtexture of the skin; increased elasticity of the skin; decreased wrinkleformation and increased endogenous elastin production in the skin,increased firmness and resiliency of the skin.

The terms “mimetic,” “peptide mimetic” and “peptidomimetic” are usedinterchangeably herein, and generally refer to a peptide, partialpeptide or non-peptide molecule that mimics the tertiary bindingstructure or activity of a selected native peptide or protein functionaldomain (e.g., binding motif or active site). These peptide mimeticsinclude recombinantly or chemically modified peptides, as well asnon-peptide agents such as small molecule drug mimetics, as furtherdescribed below.

The term “dimer”, as in a peptide “dimer”, refers to a compound in whichtwo peptide chains are linked; generally, although not necessarily, thetwo peptide chains will be identical and are linked through a linkingmoiety covalently bound to the terminus of each chain.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, rash, or gastric upset.In a preferred embodiment, the therapeutic composition is notimmunogenic when administered to a human patient for therapeuticpurposes.

“Providing” when used in conjunction with a therapeutic means toadminister a therapeutic directly into or onto a target tissue or toadminister a therapeutic to a patient whereby the therapeutic positivelyimpacts the tissue to which it is targeted. Thus, as used herein, theterm “providing”, when used in conjunction with compositions comprisingone or more manganese salts, can include, but is not limited to,providing compositions comprising one or more divalent manganese basedcompounds, trivalent iron based compounds or salts thereof into or ontothe target tissue; providing compositions systemically to a patient by,e.g., intravenous injection whereby the therapeutic reaches the targettissue; providing compositions in the form of the encoding sequencethereof to the target tissue (e.g., by so-called gene-therapytechniques).

Unless otherwise indicated, the term “skin” means that outer integumentor covering of the body, consisting of the dermis and the epidermis andresting upon subcutaneous tissue.

As used herein, the term “therapeutic” means an agent utilized to treat,combat, ameliorate, prevent or improve an unwanted condition or diseaseof a patient. In part, embodiments of the present invention are directedto improve the functionality, the appearance, the elasticity, and/or theelastin content of mammalian tissue. As it applies to skin, it ismeasured by elasticity, turgor, tone, appearance, degree of wrinkles,and youthfulness. As it applies to smooth muscle cells, blood vessels,it is measured by increased elasticity (elastin/elastic fiber synthesisand deposition) and decreased neointimal thickening (smooth muscle cellproliferation). The methods herein for use contemplate prophylactic useas well as curative use in therapy of an existing condition.

The terms “therapeutically effective” or “effective”, as used herein,may be used interchangeably and refer to an amount of a therapeuticcomposition embodiments of the present invention—e.g., one comprisingone or more plant-derived peptides. For example, a therapeuticallyeffective amount of a composition comprising plant derived peptides is apredetermined amount calculated to achieve the desired effect, i.e., toeffectively promote elastin production, collagen production, cellproliferation, or improved appearance, or improved tissue elasticity inan individual to whom the composition is administered. The tissue inneed of such therapeutic treatment may present lines or wrinkles, sundamaged tissue, or scar tissue.

The term “tissue” refers to any aggregation of similarly specializedcells which are united in the performance of a particular function. Asused herein, “tissue”, unless otherwise indicated, refers to tissuewhich includes elastin as part of its necessary structure and/orfunction. For example, connective tissue which is made up of, amongother things, collagen fibrils and elastin fibrils satisfies thedefinition of “tissue” as used herein. Additionally, elastin appears tobe involved in the proper function of blood vessels, veins, and arteriesin their inherent visco-elasticity. Thus, “tissue” thus includes, but isnot limited to skin fibroblasts and smooth muscle cells including humanaortic smooth muscle cells.

The term “unit dose” when used in reference to a therapeutic compositionof the present invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,excipient, carrier, or vehicle.

For simplicity and illustrative purposes, the principles of theinvention are described by referring mainly to an embodiment thereof. Inaddition, in the following descriptions, numerous specific details areset forth in order to provide a thorough understanding of the invention.It will be apparent however, to one of ordinary skill in the art, thatthe invention may be practiced without limitation to these specificdetails. In other instances, well known methods and structures have notbeen described in detail so as not to unnecessarily obscure theinvention.

According to one embodiment, a composition of the invention comprises asextapeptide as described herein. According to another embodiment, acomposition of the invention comprises a linked sextapeptide asdescribed herein. A composition of the invention may, alternatively,comprise a peptide mimetic of the sextapeptides or linked sextapeptidesof the invention. The invention comprises, in other embodiments,chemically digested plant extracts, as described herein.

Compositions of the present invention may stimulate the synthesis of oneor more of the extracellular matrix components by fibroblasts and othermatrix supporting cells. For example, compositions of the invention maystimulate the synthesis of extracellular matrix components such as, butnot limited to, fibrillin I, a major scaffold component of elastin,collagen type I, II, and III, fibronectin chondroitin sulfate-containingglycosaminoglycans, elastin, and lysyl oxidase. Additionally,compositions of the present invention may stimulate cell proliferationand elastin production in smooth muscle cells.

As further described herein, the compositions of the invention mayprovide the appearance of increased elastogenesis in a tissue. Oneembodiment of the present invention includes therapeutic compositionsand cosmetic preparations useful in improving or enhancing theappearance of skin comprising the aforementioned syntheticsextapeptides, or peptide mimetics thereof, or chemically digested plantextracts. The compositions of the present invention may further improvefacial lines and wrinkles, for example, through induction of newconnective tissue synthesis in skin. The compositions are used,according to another embodiment, for the restoration of cutaneousconnective tissue proteins in the skin.

In an embodiment of the present invention, compositions may beformulated into a cosmetic skin care product or cosmetic preparation toaid or facilitate the assembly of new elastic fibers in skin. Othersuitable formulations include fibroblast injections for the improvementof facial lines and wrinkles Patient dermal fibroblasts are cultured invitro and re-introduced via injection into sites presenting visiblelines and wrinkles.

The invention includes methods of using the compositions of theinvention. In one embodiment, a method of stimulating the proliferationand migration of dermal fibroblasts and elastogenesis comprisingadministering a composition of the invention in an amount sufficient toprovide the appearance of increased elastogenesis is provided. Inanother embodiment, a method of administering a composition of theinvention in an amount sufficient to stimulate elastogenesis isprovided.

Further embodiments include methods of treating premature aging,including wrinkling and folding of the skin by administeringtherapeutically effective amounts of a composition of the invention.Embodiments further include methods of treating elastin or geneticabnormalities affecting elastic fibers in skin, including, but notlimited to, Costello Syndrome, Cutis Laxa and Pseudoxanthoma Elasticum,by administering therapeutically effective amounts of a composition ofthe present invention.

As described herein, we have shown that the compositions of theinvention stimulate elastogenesis in cultures of stromal cells isolatedfrom human heart. Therefore, the invention further includes uses of thecompositions in the treatment of cardiovascular disorders that maybenefit from stimulated elastogenesis. For example, the inventioncomprises methods of using the compositions of the invention tostimulate elastic fiber formation in the scars formed after heartinfarcts. In the majority of patients with post-infarct scars, the scarsformed contain mostly collagen fibers that are stiff and non resilient.Stimulation of local stromal cells in post-infarct heart to produceelastic fibers decreases the possibility of collagenous fibrosis andcreates a stronger and more durable scar, more likely to comply withcontractile heart muscle. The compositions of the invention, wheninjected in to hypoxic heart tissue, will not effect levels of localoxygen. The present invention, therefore, provides an advantage overother experimental protocols in which injections of millions ofallogenic cells (e.g., CHO cells transfected with an elastin gene)compete for oxygen with surviving heart cells and may deteriorate nethealing of post-infarct heart.

Elastin

Elastin is secreted by fibrocytes of the connective tissues into theintercellular network. In the dermal connective tissue, the elastinfibers are thin and sinuous. Elastin contained in the dermis represents5% of its dry weight. Elastin is a large fibrous protein which is formedby spiral filaments that can be compared to springs. The spiralfilaments consist of peptidic chains that can stretch out. The peptidicchains are connected to each other by very specific amino-acids:desmosin and isodesmosin, which builds between them, giving the moleculea reticular aspect. After stretching out, the molecules resume theiroriginal shape due to this cross linking, which is essential tomolecular elasticity.

The biosynthesis of elastin begins with the embryonic period andcontinues through adulthood, at which time the body stops producingelastin. Thus, elastin is no longer renewed. With aging, the elasticfibers progressively degenerate and separate into fragments. The skinprogressively loses its elasticity, resulting in fine lines andwrinkles. This damage to our elastic tissue cannot be avoided and ispart of the natural (physiological) aging process. This process beginsrelatively early, but accelerates considerably after age 40.

Wound healing is a complex biological process that involves manydifferent cell types, many different cytokines, the extracellular matrix(ECM), and numerous interactions among them. Most wounds heal rapidlyand efficiently within a week or two; however, the result is neitheraesthetically nor functionally perfect. Wound contraction and scarformation are currently unavoidable results of wound healing. Scartissue is less flexible than normal skin and can be cosmeticallydisfiguring, and wound contraction can lead to joint disablement. Scarslack elastin and consist of a poorly reconstituted collagen matrix indense parallel bundles rather than the mechanically efficientbasket-weave meshwork of collagen in the unwounded dermis.

As wounds heal a new stratified epidermis is reestablished from themargins of the wound inward. Matrix formation and remodeling beginssimultaneously with re-epithelialization. The matrix is constantlyaltered over the next several months with the elimination of thefibronectin from the matrix and the accumulation of collagen thatprovides the residual scar with increasing tensile strength. Elastinfibers, which are responsible for the elasticity of tissue, are onlydetected in human scars years after the injury.

Many methods have been proposed and tested to promote wound healing andlimit scarring; however, better methods and compositions are stillneeded. Wounds that can be treated with the compositions of theinvention include, but are not limited to, cutaneous wounds, cornealwounds, and injuries to the epithelial-lined hollow organs of the bodyand post-infarct heart. Wounds suitable for treatment include thoseresulting from trauma such as burns, abrasions and cuts, decubitus andnon-healing varicose and diabetic ulcers, as well as wounds resultingfrom surgical procedures such as incisions and skin grafting. Accordingto one embodiment, the compositions of the invention, for example,chemically digested plant extracts, can be used to treat infected woundsand ulcers, in which proteolytic enzymes released from dying cells andbacteria can facilitate further cleavage of peptides within apreparation and release smaller elastogenic peptides containingEBPL-like domains.

Stimulation of the deposition of elastin at the site of injury bycomposition of the invention will aid in promoting wound healing whilelimiting scarring. Initially, the stimulated deposition of elastin willhold the injured tissue together. The stimulation of elastin synthesisby a composition of the invention according to some embodiments willfurther act as a chemotactic attractant for fibroblasts, endothelialcells, and inflammatory cells, which will promote healing of the injuredtissue. Elastin synthesis at the site of injury will also lessenscarring since scar tissue is devoid of elastin, and elastin is animportant component of uninjured skin. The stimulation and secretion ofelastin into the matrix will also generally provide a favorableenvironment for the cells that participate in the healing process,further accentuating the wound healing process.

A composition of the invention, according to one embodiment, willstimulate the migration of fibroblasts into the treated area. Accordingto another embodiment, a composition of the invention will interact withelastin receptors on the fibroblasts and stimulate the secretion ofelastin (i.e., elastogenesis). In one embodiment, a composition of theinvention is applied to the wound and maintained in contact with thewound for an extended period of time, i.e., during the entire healingprocess or until at least closure of the wound occurs by new tissue.

Elastin owes its properties to its thin structure which resembles thatof rubber. Elastin is the protein responsible for our skin's essentialelasticity and tonicity. Its decrease means the skin starts sagging,allowing fine lines, folds and wrinkles to appear and grow. The presentinvention provides compositions comprising a sextapeptide compound, alinked sextapeptide, a peptide mimetic of either of these, or chemicallydigested rice bran, as a stimulator of elastogenesis. This occurs,according to some embodiments, by the composition of the inventioninteracting with elastin receptors and stimulating the proliferation andmigration of dermal fibroblasts and other supporting cells to an area ofskin in need of elastogenic properties. Such areas of skin include, butare not limited to, wounds, sagging and/or wrinkled skin, stretchedskin, skin damaged by UV or other radiation or by environmental damage,etc. The compositions of the invention induce elastogenesis in dermalfibroblasts, and by their interaction with the elastin receptors, causecells to increase the secretion of insoluble elastin fibers into theextracellular matrix. Thus, the present invention provides compositionsand methods to compensate for the loss of elastic components in thedermis.

The result of aging on skin, whether or not it has been accelerated byenvironmental damage (such as radiation, pollution, etc.), is adeterioration of the dermal layer: fewer fibroblasts, less collagen,less elastin and less circulatory support. Consequently, normalstretching and contraction of the skin leads to damage of the dermisthat is not readily corrected, resulting in wrinkling and/or sagging.The present invention provides methods and compositions for stimulatingthe proliferation and migration of dermal fibroblasts into an area inneed of elastogenic properties. According to some embodiments, thecompositions of the invention interact with elastin receptors onfibroblasts and stimulate the secretion of elastin. The enhanced elastindeposition reduces the effects of radiation (for example, but notlimited to, ultraviolet radiation) or other environmental damage byproviding elasticity and tone to the skin.

Elastogenic Peptides, or Peptide Mimetics Thereof

In accordance with one embodiment of the present invention, novelpeptides derived from or based on protein sequences found in plants areprovided. The compositions of the invention according to one embodimentprovide the appearance of increased elastogenesis. In other embodiments,the compositions stimulate elastogenesis and migration of dermalfibroblasts. In one embodiment, such stimulation is the result of theinteraction of the composition of the invention with elastin receptors.In one embodiment, a synthetic sextapeptide is provided, which binds toelastin receptors and stimulates elastogenesis. The peptides of theinvention are also referred to herein as elastin binding proteinligands, or EBPLs.

In an embodiment of the invention, the sextapeptide comprises thesequence X₁-X₂-X₃-X₄-X₅-X₆ (SEQ ID NO. 3), wherein X₁ is V or I, X₂ isG, X₃ is A or L, X₄ is M or S, X₅ is P and X₆ is G. In one embodiment,the sextapeptide of the invention comprises VGAMPG (SEQ ID NO. 4),VGLSPG (SEQ ID NO. 5), IGAMPG (SEQ ID NO. 6), or IGLSPG (SEQ ID NO. 7).

In further embodiments, a linked sextapeptide (or sextapeptide dimer) isprovided wherein two sextapeptides of the invention are joined with oneor more additional linking amino acid residues (a “linking moiety”).According to one embodiment, the synthetic linked sextapeptide has thesequence X₁-X₂-X₃-X₄-X₅-X₆, -X₇-X₁-X₂-X₃-X₄-X₅-X₆ (SEQ ID NO. 16),wherein X₇ is a linking moiety; X₁ is independently selected from V andI; X₂ is G; X₃ is independently selected from A and L; X₄ isindependently selected from M and S; X₅ is P; and X₆ is G. The linkingmoiety of a linked sextapeptide can be any moiety recognized by thoseskilled in the art as suitable for joining the sextapeptides so long asthe sextapeptide compound(s) retain the ability to interact with theelastin receptor and induce elastogenesis. The linking moiety may becomprised of, for example, but not limited to, at least one of alanineor any other amino acid, a disulfide bond, a carbonyl moiety, ahydrocarbon moiety optionally substituted at one or more availablecarbon atoms with a lower alkyl substituent. Optimally, the linkingmoiety is a lysine residue or lysine amide, i.e., a lysine residuewherein the carboxyl group has been converted to an amide moiety —CONH.

The compositions of the invention may also comprise linked sextapeptideswherein the two sextapeptides that are joined by a linker are differentsextapeptides.

In one embodiment, the linked sextapeptide of the invention comprisesVGAMPGAAAAAVGAMPG (SEQ ID NO. 8), VGLSPGAAAAAVGLSPG (SEQ ID NO. 9),VGVAPGAAAAAVGVAPG (SEQ ID NO. 10), IGAMPGAAAAAIGAMPG (SEQ ID NO. 11), orIGLSPGAAAAAIGLSPG (SEQ ID NO. 12). Also provided by the invention is alinked sextapeptide comprising the sequence IGVAPGAAAAAIGVAPG (SEQ IDNO. 14). Another linked sextapeptide of the invention comprises thesequence of two sextapeptides having the sequence IGVAPG (SEQ ID NO. 13)that are joined by a linker, as described above.

The present invention also provides non-peptide or partial peptidemimetics of any of the aforementioned synthetic peptides. According toanother aspect of the invention, a compound that binds to elastinreceptors, stimulates migration of dermal fibroblasts and additionallystimulates fibroblast elastogenesis is provided. The compound has aformula X₁-X₂-X₃-X₄-X₅-X₆ (SEQ ID NO. 17), wherein X₁ is V or I or amimetic of V or I; X₂ is G, or a mimetic of G; X₃ is A or L, or amimetic of A or L; X₄ is M or S, or a mimetic of M or S, X₅ is P or amimetic of P and X₆ is G or a mimetic of G. In alternative embodiments,the compound may comprise the formula X₁-X₂-X₃-X₄-X₅-X₆-X₇ (SEQ ID NO.18), wherein X₇ is one or more linking amino acid residues comprisingalanine wherein the linking residues join two sextapeptide compounds toeach other, each sextapeptide having the sequence X₁-X₂-X₃-X₄-X₅-X₆ (SEQID NO. 17), wherein X₁ is V or I or a mimetic of V or I; X₂ is G, or amimetic of G; X₃ is A or L, or a mimetic of A or L; X₄ is M or S, or amimetic of M or S, X₅ is P or a mimetic of P and X₆ is G or a mimetic ofG.

A further embodiment of the present invention relates to peptidemimetics of GXXPG peptides. In one embodiment, the peptides of theinvention are modified to produce peptide mimetics by replacement of oneor more naturally occurring side chains of the 20 genetically encodedamino acids (or D amino acids) with other side chains, for instance withgroups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7 memberedalkyl, amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy,hydroxy, carboxy and the lower ester derivatives thereof, and with 4-,5-, 6-, to 7 membered heterocyclics. For example, proline analogs can bemade in which the ring size of the proline residue is changed from 5members to 4, 6, or 7 members. Cyclic groups can be saturated orunsaturated, and if unsaturated, can be aromatic or nonaromatic.Heterocyclic groups can contain one or more nitrogen, oxygen, and/orsulphur heteroatoms. Examples of such groups include the furazanyl,furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl,isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g.1-piperazinyl), piperidyl (e.g. 1-piperidyl, piperidino), pyranyl,pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl,pyrimidinyl, pyrrolidinyl (e.g. 1-pyrrolidinyl), pyrrolinyl, pyrrolyl,thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g. thiomorpholino),and triazolyl. These heterocyclic groups can be substituted orunsubstituted. Where a group is substituted, the substituent can bealkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.Peptidomimetics may also have amino acid residues that have beenchemically modified by phosphorylation, sulfonation, biotinylation, orthe addition or removal of other moieties.

A variety of techniques are available for constructing peptide mimeticswith the same or similar desired biological activity as thecorresponding native but with more favorable activity than the peptidewith respect to solubility, stability, and/or susceptibility tohydrolysis or proteolysis. See, e.g., Morgan & Gainor, Ann. Rep. Med.Chem. 24:243-252 (1989). Certain peptidomimetic compounds are based uponthe amino acid sequence of the peptides of the invention. Often,peptidomimetic compounds are synthetic compounds having athree-dimensional structure (i.e. a “peptide motif”) based upon thethree-dimensional structure of a selected peptide. The peptide motifprovides the peptidomimetic compound with the desired biologicalactivity, i.e., binding to PIF receptors, wherein the binding activityof the mimetic compound is not substantially reduced, and is often thesame as or greater than the activity of the native peptide on which themimetic is modeled. Peptidomimetic compounds can have additionalcharacteristics that enhance their therapeutic application, such asincreased cell permeability, greater affinity and/or avidity andprolonged biological half-life.

Peptidomimetic design strategies are readily available in the art. See,e.g., Ripka & Rich, Curr. Op. Chem. Biol. 2:441-452 (1998); Hruby etal., Curr. Op. Chem. Biol. 1:114-119 (1997); Hruby & Balse, Curr. Med.Chem. 9:945-970 (2000). One class of peptidomimetics a backbone that ispartially or completely non-peptide, but mimics the peptide backboneatom-for atom and comprises side groups that likewise mimic thefunctionality of the side groups of the native amino acid residues.Several types of chemical bonds, e.g., ester, thioester, thioamide,retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, areknown in the art to be generally useful substitutes for peptide bonds inthe construction of protease-resistant peptidomimetics. Another class ofpeptidomimetics comprises a small non-peptide molecule that binds toanother peptide or protein, but which is not necessarily a structuralmimetic of the native peptide. Yet another class of peptidomimetics hasarisen from combinatorial chemistry and the generation of massivechemical libraries. These generally comprise novel templates which,though structurally unrelated to the native peptide, possess necessaryfunctional groups positioned on a nonpeptide scaffold to serve as“topographical” mimetics of the original peptide (Ripka & Rich, 1998,supra).

Chemically Digested Rice Bran Extracts

According to one embodiment, chemically digested plant extracts areprovided that stimulate or appear to stimulate elastogenesis in atissue. In one embodiment, such plant extracts are obtained from ricebran. The chemically digested rice bran extracts described herein werefound to be immuno-reactive with a panel of antibodies raised to humantropoelastin. Furthermore, chemical digests of both soluble andinsoluble rice bran contained the unique crosslinking amino acid,desmosine (1742/mg protein and 1638/mg protein respectively). Thesecharacteristics suggest the presence of one or more elastin-likepeptides in rice bran. Thus, the present invention comprises suchelastin-like peptide preparations, including pharmaceutical compositionsand cosmetic preparations, and chemically digested rice branpreparations, as well as methods of using compositions comprising suchpreparations to treat the conditions discussed herein.

Pharmaceutical Compositions

In embodiments of the present invention, the compositions of theinvention may be formulated into pharmaceutical compositions. Forexample, in one embodiment, topical carriers may be employed whichshould be both non-irritating to the skin and which are suitable fordelivering the active components to the skin. Further, suitable topicalcarriers should be those which do not inhibit the antioxidant activityof the active ingredients thus reducing the efficiency of thecomposition for protecting the skin from the effects of free radicaldamage which can occur due to, for example, acute and chronicultraviolet radiation. Further, such carriers must be of sufficientlyhigh purity and sufficiently low toxicity to render them suitable forchronic topical administration to the skin and be free of bacterialcontaminants.

The compositions of the invention described herein can be incorporatedinto any suitable pharmacologically acceptable carrier which is suitablefor topical administration to the human skin. As such, thepharmacologically acceptable carrier must be of sufficient purity andhave sufficiently low toxicity to render it suitable for administrationto a human noting that, generally, the carrier can represent up to99.99% and typically from at least approximately 80% of the totalcomposition. Thus, the phrase “pharmaceutically acceptable” refers tomolecular entities and compositions that do not produce an allergic orsimilar untoward reaction when administered to a human. Thepharmaceutically acceptable carriers and additives employed in thepresent compositions are compatible with the compositions of theinvention.

Typical compositions for use herein include a wide variety of physicalforms. These include, but are not limited to, solutions, lotions,creams, oils, gels, sticks, sprays, ointments, balms, patches andpastes. Generally, such carrier systems can be described as beingsolutions, creams, emulsions, gels, solids and aerosols.

Solvents are generally employed in the preparation of suitable topicalcompositions. Such solvents can either be aqueous or organic based and,in either case, the solvent must be capable of having dispersed ordissolved therein the above-described active components while not beingirritating to the user. Water is a typical aqueous solvent whilesuitable organic solvents include propylene glycol, battalion glycol,polyethylene glycol, polypropylene glycol, glycerol, 1,2,4-butanetriol,sorbitol esters, 1,2,6-hexanetriol, ethanol, isopropanol, butanediol andmixtures thereof. Solvents can be included in the overall composition inamounts ranging from 0.1% to 99% and preferably from 2.0% to 75%. It isnoted that compositions of the present invention can be produced in theform of an emollient. A wide variety of suitable emollients are knownand may be used herein. In this regard, reference is made to U.S. Pat.No. 5,296,500, the disclosure of which is hereby incorporated byreference.

Alternatively, the present composition can be formulated as a lotioncontaining from about 0.0002% to about 10% by weight of a peptidecomposition of the invention, e.g. a sextapeptide of the invention, alinked sextapeptide of the invention, or a peptide mimetic of thesextapeptides or linked sextapeptides of the invention. Alternatively, alotion can be formulated containing about 1-10 mg/mL rice bran extract,prepared, for example, by any of the methods described herein. Lotionsand creams can be formulated as emulsions as well as solutions. Further,the product can be formulated from a solution carrier system as a cream.A cream comprising a composition of the present invention wouldpreferably comprise from about 0.1% to 15% and preferably from 1% to 5%active ingredients. Final products may contain up to 10% by weight butpreferably 0.001 to 5% of an active ingredient, though of course moreconcentrated or more dilute solutions may also be used in greater orlesser amounts. For example, an eye cream may comprise about 0.0012%(w/w) and a facial cream may comprise about 0.0003% (w/w) of asextapeptide compound, a linked sextapeptide compound or peptide mimeticthereof, in an excipient.

The peptide compositions of the invention (0.0004%-0.002%) and plantextracts of the invention (about 1-10 mg/ml), in a suitable cream,lotion or ointment base, may be applied topically to help restoreelastic fibers in aged, sun damaged or wounded skin.

Peptide compositions of the invention (0.0004%-0.002%) and plantextracts of the invention (about 1-10 mg/ml), in a suitable cream,lotion or ointment base, may be applied topically to dilate existingcutaneous small blood vessels for the purpose of healing decubitus ordiabetic ulcers that result from the contraction of small vesselssupplying blood to the skin.

The peptides of the invention (10-20 μg/mL) or plant extracts of theinvention (1-10 mg/ml) may be injected into coronary arteries duringballoon angioplasty, injected intravenously, or injected directly intomyocardial post-infarct scar tissue during open hear surgery as a meansof stimulating new elastogenesis that would lend strength and resiliencyto the post-infarct scar of the human heart.

Both crude and chemically purified plant extracts or preparations can beused in topical application for healing of decubitus or diabetic ulcersthat result from the contraction of small vessels supplying blood to theskin. Because sequential oxalic acid or trypsin digestion of rice branreduced the molecular weight of peptides in rice bran preparations, wehypothesize that even large and insoluble elastin-like proteins will beeventually partially digested by enzymes released from dead cells andbacteria (always contaminating those open skin ulcerations) and deliverthe desirable peptide that would stimulate relaxation and opening ofcontracted capillaries and stimulate production of new extracellularmatrix rich of new elastin.

Manganese salts (MnCl₂, MnSO₄ and MnaPCA) and trivalent iron (FerricAmmonium Citrate, FAC) have each been shown to individually stimulatethe production and assembly of new tropoelastin into new elastic fibers.The compositions of the present invention may be formulated to furtherinclude a manganese component and/or a trivalent iron component.Additionally, compounds comprising sodium are suitable additives fortherapeutic compositions of the present invention. Sodium has beenlinked to the stimulation of elastogenesis. Copper, an activator oflysyl oxidase (an enzyme that crosslinks tropoelastin molecules intoinsoluble polymeric elastin) is another suitable additive used in thetherapeutic compositions of the present invention.

Optionally, a manganese component may be added to a composition of theinvention. The manganese may be any manganese compound, or apharmaceutically acceptable salt thereof, but preferably is MnCl₁, MnSO₄and/or MnPCA, wherein the manganese component is typically present in anamount from about 0.5 to 10 weight percent, preferably from about 1 to 8weight percent and most preferably from about 5 to 7 weight percent,wherein the manganese is present in an amount from about 5 to 20 weightpercent of a complex.

Optionally a trivalent iron component (such as, but not limited to,Ferric Ammonium Chloride (FAC) may also be included in thepharmaceutical composition. The trivalent iron component stimulates newelastogenesis and assists in treatment of elastic tissue defects. Thetrivalent iron, when included in the composition, is generally presentin an amount from about 5 to 20 weight percent. In one embodiment thetrivalent iron component is generally present in an amount from about0.01 to 5 weight percent, preferably from about 0.02 to 3 weightpercent, and more preferably from about 0.03 to 2 weight percent of thecomposition.

Optionally, a sodium component, or pharmaceutically acceptable saltthereof, may also be included in a pharmaceutical composition of theinvention. The sodium component is generally present in about 5 to 20percent of the complex. The sodium component may generally be present inan amount of about 1 to 10 percent weight percent, or from about 5 to 7percent weight of the composition.

A copper component may also be included in the pharmaceuticalcomposition, and may be any copper compound or pharmaceuticallyacceptable salt thereof. The copper component inhibits elastase andassists in the treatment of elastic tissue defects. The copper compoundmay be in the form of copper sebacate. When included in a compositionthe copper component is generally present in an amount of about 5 to 20weight percent of the copper compound, such as copper sebacate. Thecopper component is generally present in an amount of about 0.01 to 5percent weight or from about 0.03 to 2 percent weight of thecomposition.

It is contemplated that as one embodiment, the active ingredientsdescribed above be used as a lotion or cream emulsion of theoil-in-water type or as a water-in-oil type, both of which beingextremely well known in the cosmetic field. Multi-phase emulsions suchas the water-in-oil type is disclosed in U.S. Pat. No. 4,254,105, thedisclosure of which is incorporated herein by reference.

It is further contemplated that the active ingredients of the presentinvention be formulated from a solution carrier system as an ointment.An ointment may comprise a simple base of animal or vegetable oils orsemi-solid hydrocarbons (oleaginous). Ointments may also compriseabsorption ointment bases which absorb water to form emulsions. Ointmentcarriers may also be water soluble. An ointment may comprise from 1% to99% of an emollient plus to about 0.1% to 99% of a thickening agent.Reference is again made to U.S. Pat. No. 5,296,500 and the citationscontained therein for a more complete disclosure of the variousointment, cream and lotion formulations for use herein.

The present composition can include one or more of a variety of optionalingredients, such as, but not limited to, anti-inflammatory agents,sunscreens/sunblocks, stimulators of protein synthesis, cell membranestabilizing agents (i.e., carnitine), moisturizing agents, coloringagents, opacifying agents and the like, so long as they do not interferewith the elastin receptor binding properties of the sextapeptidecompounds, or peptide mimetics thereof.

Additional components of the therapeutic compositions may include anysuitable additive that has been used in cosmetics or other skin carecompositions. These include, but are not limited to aloe vera,antioxidants, azulene, beeswax, benzoic acid, beta-carotene, butylstearate, camphor, castor oil, chamomile, cinnamate, clay, cocoa butter,coconut oil, cucumber, dihydroxyacetone (DHA), elastin, estrogen,ginseng, glutamic acid, glycerin, glycolic acid, humectant,hydroquinone, lanolin, lemon, liposomes, mineral oil, monobenzone,nucleic acids, oatmeal, paba, panthenol, petroleum jelly, propeleneglycol, royal jelly, seaweed, silica, sodium lauryl sulfate sulfur,witch hazel, zinc, zinc oxide, copper, hyaluronic acid and shea butter.

The compositions comprising the synthetic sextapeptide, synthetic linkedsextapeptides, peptide mimetic thereof, or chemically digested plantextracts may further comprise retinoic acid, excipients, or otheradditives. For example, retinoic acid acts to stimulate collagenproduction and may be useful as an additive to compositions of thepresent invention.

Additives which aid in improving the elasticity of elastin comprisingtissues such as tretinoin, vitamin E, sources of copper, and/ormagnesium ions, retinol, copper peptides, and any one of the 20 standardamino acids may also be added to the compositions of the presentinvention. Additives which induce deposition of tropoelastin onmicrofibril scaffolds, and compounds which induce lysyl oxidaseactivity, such as transforming growth factor beta-1 and copper (supra),may also be added to such compositions. Compositions of the presentinvention may include a therapeutically and biologically compatibleexcipient.

The formulation can also include other active ingredients, such asantibiotics, analgesics, anti-allergenics and the like. The formulationis commonly applied to the skin as a lotion or cream to be rubbed onbody tissue over the desired area. For optimum efficacy treatment inaccordance with the presented method should be initiated as early aspossible following exposure to sunlight, or other radiation source, orto skin injury (wound, etc). The formulation is generally applied to theskin once or twice daily. As noted elsewhere herein, the presentcomposition may also be used to inhibit and/or minimize the effects ofaging and/or photo damage on the skin, as well as to prevent and/ortreat scars (such as cutaneous hypertrophic scars).

Administration

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as direct topical application, application via atransdermal patch and the like.

For topical administration in an aqueous solution, for example, thecompositions of the invention may be used directly on the skin withoutany toxic effects to the patient. Alternatively, the compositions of theinvention may be dissolved or resuspended in a suitable buffer prior tomixing, if necessary.

In general, routine experimentation will determine specific ranges foroptimal therapeutic effect for each composition and each administrativeprotocol, and administration to specific individuals will be adjusted towithin effective and safe ranges depending on the condition andresponsiveness of the individual to initial administrations.

Some variation in dosage will necessarily occur depending on thecondition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Moreover, for human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA.

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts whichcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention. Various modifications and combinations of the illustrativeembodiments, as well as other embodiments of the invention, will beapparent to persons skilled in the art upon reference to thedescription.

The compositions of the present invention induce synthesis anddeposition of elastin and induce cellular proliferation in normal humandermal fibroblasts. The following effects in culture compositions arebetter understood in reference to the examples below.

Example 1

Reagents

Soluble and insoluble rice bran were obtained from CGS Group, Inc.VGVAPG was obtained from Sigma (St. Louis, Mo.). EBPL-1(VGVAPGAAAAAVGVAPG) (SEQ ID NO. 10), EBPL-2 (IGVAPG) (SEQ ID NO. 13) andEBPL-3 (IGVAPGAAAAAIGVAPG) (SEQ ID NO. 14) were organically synthesizedaccording to our design by EZBIOLAB, Inc. All peptides were synthesizedat 95% purity.

Preparation of Chemically Digested Soluble Rice Bran.

One gram of Soluble Rice Bran (Nutri Select Soluble—Nutri Rice CDG GroupInc.) was suspended in 40 ml of 1M NaOH diluted in 80% ethanol andincubated for one hour. The preparation was then centrifuged at 4000 rpmfor 10 min, the pellet was re-suspended in 20 ml of distilled water,dialyzed (MWCO 2000) overnight against distilled water and thanlyophilized. The product was then re-suspended in 5 ml of 0.25M oxalicacid and boiled for 1 hour. After centrifugation at 4000 rpm for 10 minthe supernatant No 1 was collected and remaining pellet was re-suspendedagain in 5 ml of 0.25M oxalic acid and boiled. This procedure wasrepeated three times until the entire material was dissolved. Allsubsequent supernatants were combined with supernatants No 1 anddialyzed against 5 changes of distilled water and then lyophilized. Theresulting 130 mg of white water soluble powder (the chemically digestedrice bran—CDRB) contained 5 mg pure protein. Alternatively, the ricebran samples were also digested with amylase (to remove starch) and thentreated with Cyanogen Bromide (CNBr) to solubilize non-crosslinkedproteins.

Western Blots.

Both soluble and insoluble rice bran were filtered through a 3,000 Dafiltration membrane and 20 μg samples of each were separated by SDS-PAGEelectrophoresis and transferred to nitrocellulose. The nitrocellulosewas subjected to western blotting with anti elastin, anti-VGVAPG (BA4)antibody, and antibody raised to AKAAAKAAAKA (SEQ ID NO. 15) (domain ontropoelastin that may be engaged in formation of crosslinking desmosinesfrom four lysines). See Starcher, B, Conrad, N., Hinek, A. and Hill, C.H., Connect. Tissue Res. 40:273-282 (1999).

Cell Cultures.

Biological effects of soluble rice bran, insoluble rice bran, VGVAPG,EBPL-1, EBPL-2 and EBPL-3 were tested in cultures of skin fibroblastsderived from healthy Caucasian females of different ages. Biologicaleffects of chemically digested rice bran (CDRB) was tested in culturesof skin fibroblasts derived from punch biopsies of healthy skin from37-year old Caucasian female. All fibroblasts were originally isolatedby allowing them to migrate out of skin explants and then passaged bytrypsinization and maintained in alpha-minimum essential mediumsupplemented with 20 mM Hepes, 1% antibiotics and antimycotics, 1%L-Glutamate and 2% fetal bovine serum (FBS). In all experiments exceptthose using CDBR, consecutive passages 3-7 were tested. Cells wereincubated in the presence and absence of VGVAPG (SEQ ID NO. 1) (5μg/mL), EBPL-2 (5 μg/mL) and EBPL-3 (10 μg/mL). In experiments usingCDRB cell passage 3 was tested, cells were densely plated (50×10⁶cells/dish) to rich immediate confluency and than cultured for 7 days inthe presence and absence of 25 μg/ml CDBR (added at day 1 and 3).

Assessment of Deposition of Elastic Fibers.

7-day-old confluent cultures of fibroblasts, which produce abundant ECM,were used. All cultures were fixed in cold 100% methanol at −20° C. for30 min, blocked with 1% normal goat serum (NGS) and then incubated for 1hour with 2 μg/ml of polyclonal antibodies to tropoelastin. All cultureswere then incubated for an additional hour with appropriatefluorescein-conjugated secondary antibodies (GAR-FITC). Nuclei werecounterstained with propidium iodide. Morphometric analysis of culturesimmunostained with antibodies recognizing extracellular matrixcomponents was performed using a computerized video analysis system(Image-Pro Plus software 3.0, Media Cybernetics, Silver Spring, Md.) asdescribed previously. Means and standard deviations were calculated andstatistical analyses were carried out by ANOVA.

Results and Discussion.

Western blots with polyclonal antibody to tropoelastin and monoclonalantibody BA4, recognizing VGVAPG (SEQ ID NO. 1) sequence on elastin andanother sterically similar GXXPG (SEQ ID NO. 2) hydrophobic domains,indicated that both soluble and insoluble rice brans containtropoelastin-like epitopes and other GXXPG (SEQ ID NO. 2) sequences.Additional immunoblotting with antibody raised to AKAAAKAAAKA alsoindicated that this domain, unique to tropoelastin, is also present inthe chemically digested rice bran preparations (FIG. 10). Based on theseand other results, the rice bran preparations of the invention appear toinclude an elastin-like peptide.

Incubating the filtered (<3,000 Da) soluble and insoluble rice bran withdermal fibroblasts revealed that both these preparations can inducedeposition of elastic fibers in vitro (FIG. 1). FIG. 1 presents primarycultures of human dermal fibroblasts, derived from a 44 year-old female,maintained for 7 days in the presence or absence of soluble or insolublerice bran (<3,000 Da). Cells were fixed and immunostained withanti-tropoelastin antibodies and fluorescein-conjugated secondaryantibodies (GAR-FITC).

Since we have previously shown that elastin receptor (elastin bindingprotein) ligands are present in ProK-60 (bovine ligamentum nuchaeproteolytic digest) and can stimulate elastogenesis, we ran a BLASTsearch for short, nearly identical, sequences resembling VGVAPG (SEQ IDNO. 1) that might also serve as ligands for the elastin binding protein,a major component of the cell surface elastin receptor. This searchrevealed the EBPL sequences VGAMPG (SEQ ID NO. 4), VGLSPG (SEQ ID NO.5), IGAMPG (SEQ ID NO. 6), IGLSPG (SEQ ID NO. 7), and IGVAPG (SEQ IDNO. 1) in protein derived from rice (Oryza sativa). Both IGVAPG (SEQ IDNO. 13) (EBPL-2) and its duplex polymer, IGVAPGAAAAAIGVAPG (SEQ ID NO.14) (EBPL-3), were organically synthesized and then tested in primarycultures of dermal fibroblasts. We hypothesized that both of thesesynthetic peptides would spontaneously fold into three-dimensionalstructures capable of interacting with the cell surface elastinreceptor, and in turn, stimulate elastogenesis as VGVAPG (SEQ ID NO. 1)does. While not wishing to be bound by theory, it appears from ourexperiments that all synthetic EBPLs stimulated elastogenesis throughinteraction with the cell-surface elastin receptor (EBP=elastin bindingprotein) in a dose dependant manner.

Immunostaining with anti elastin antibody of 7 day-old primary culturesof dermal fibroblasts incubated in the presence of tested reagentsdemonstrated that our new synthetic peptides EBPL-2 and EBPL-3 arecapable of inducing elastogenesis to the levels that exceed thoseobtained in cultures treated with VGVAPG (SEQ ID NO. 1) or withsynthetic peptide containing its doublet VGVAPGAAAAAVGVAPG (SEQ ID NO.10) (EBPL-1) (FIG. 2). FIG. 2 presents primary cultures of human dermalfibroblasts, derived from 38 and 44 year-old females, maintained for 7days in the presence or absence of VGVAPG (SEQ ID NO. 1) (5 μg/mL),EBPL-2 (5 μg/mL) and EBPL-3 (10 μg/mL). Cells were fixed andimmunostained with anti-tropoelastin antibodies andfluorescein-conjugated secondary antibodies (GAR-FITC). Interestingly,fibroblasts stimulated with VGVAPG (SEQ ID NO. 1) and EBPL-1demonstrated very thin extracellular elastic fibers at levels greaterthan the control levels, whereas EBPL-2- and EBPL-3-treated culturesdemonstrated deposition of thick extracellular elastic fibers. Theseresults indicate that synthetic peptides reflecting the rice-derivedsequence strongly stimulate elastogenesis and justify the abovedescribed effect obtained in cultures exposed to crude protein extractsfrom rice bran.

These preliminary data suggest that both EBPL-2 and EBPL-3 (containingIGVAPG (SEQ ID NO. 13) sequences mimicking the elastin receptor-bindingdomain, detected in the elastin-like rice bran derived protein) induceelastogenesis in dermal fibroblasts through their interaction with thecell-surface elastin receptor. We propose that, in contrast to fulllength tropoelastin or high molecular constructs reflecting tropoelastinexons, the small molecular weight synthetic peptides EBPL-2 and EBPL-3(that can more easily penetrate through the epidermis) can constitutenovel biologically active ingredients that can be formulated intosuitable topical cosmetic anti-aging creams/lotions aimed at restorationof elastic fibers.

Example 2

Production of new elastic fibers was also monitored by metaboliclabeling tropoeelastin produced by cultured fibroblasts and itssubsequent incorporation into insoluble elastin, the major component ofelastic fibers. Fibroblasts were densely plated (50×105/dish) and grownto confluency in 10 cm cell culture dishes in quadruplicate. 20 μCi of[³H]-valine was added to each dish along with fresh media. Cultures werethen incubated for 72 hours and insoluble elastin was assessedseparately in each culture. After media was removed, the cell layers anddeposited extracellular matrix were scraped in 0.1N NaOH, sedimented bycentrifugation, and boiled in 0.5 mL of 0.1N NaOH for 45 minutes tosolubilize all matrix components except elastin. The resulting pelletscontaining the insoluble elastin were then solubilized by boiling in 200μL of 5.7 N HCl for 1 hour and the aliquots were mixed withscintillation fluid and counted.

FIG. 3 shows the quantitative assessment of [³H]-Valine incorporationinto newly deposited insoluble elastin in dermal fibroblast cultures.Fibroblast cultures were stimulated daily for six days withGXXPG-containing peptides alone and in combination with copper,manganese and iron salts. These results show that the addition of metalsalts enhances peptide-induced deposition of insoluble elastic fibers invitro.

FIG. 4 shows elastin deposition in 7 day-old dermal fibroblasts culturesin the presence and absence of CDRB. The CDRB treated culturessynthesize more tropoelastin (detected intracellularly) and deposit moreextracellular elastic fibers as compared to control cultures.

Example 3

Immunocytochemistry of 7 day-old fibroblast cultures withanti-tropoelastin antibody demonstrated that dermal fibroblasts derivedfrom a female patient with stretch marks produced significantly moreelastic fibers when treated with the chemically digested rice bran(CDRB). The CDRB-treated fibroblasts also demonstrated moreimmuno-detectable tropoelastin in their intracellular Golgi compartmentthan the untreated control fibroblast.

Preliminary immunocharacterization of CDRB showed immunoreactivity witha polyclonal elastin antibody, suggesting that CDRB may contain similarsecondary structures found in mammalian elastin.

Data demonstrates that the rice-based peptides are much more (>500%)potent elastogenic inducers than ProK-60. This was confirmed throughindirect immuno-fluorescence. Results were confirmed with Northern Blotsand metabolic labeling of insoluble elastin (data not shown).

Example 4

Using a BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) we identifiedthe IGVAPG (SEQ ID NO. 13) domain in the amino acid sequence for atranscription factor of Japanese rice, which also reflects the commonEBP-binding GXXPG (SEQ ID NO. 2) motif found in elastin and laminin. Weused RT-PCR to measure transcription of the elastin gene by peptides ofthe invention. FIG. 5 shows that the synthetic hexapeptide, IGVAPG (SEQID NO. 13), (Code Name: EBPL-2™) increased transcription of the elastingene as does VGVAPG (SEQ ID NO. 1) in cultures of adult dermalfibroblasts. The fact that both EBPL-2 and VGVAPG (SEQ ID NO. 1)stimulated transcription in adult dermal fibroblasts maintained inserum-free medium is indicative that both peptides are primarystimulators of elastogenesis. We also found that the observed higherlevels of elastin mRNA were indeed translated into higher levels oftropoelastin protein (monomeric precursor of elastin).

Example 5

Culturing adult dermal fibroblasts in the presence of EBPL-2 and VGVAPG(SEQ ID NO. 1) for 7 days yielded enhanced levels of intra- andextracellular tropoelastin, as confirmed by immunocytochemical stainingwith an anti-tropoelastin antibody (FIG. 6). (A) As depicted in FIG. 6(A) EBPL-2 and VGVAPG (SEQ ID NO. 1) treated adult dermal fibroblastsmaximally produced 710% and 457% more tropoelastin, respectively, thancontrol cultures as assessed by quantitative morphometric analysis ofimmuno-detectable tropoelastin. EBPL-2-treated fibroblasts maximallyproduced 141% more tropoelastin than maximum ProK-60-induced levels. (B)Representative tropoelastin immunostains of control, ProK-60-(25 μg/mL),EBPL-2-(25 μg/mL) and VGVAPG (SEQ ID NO. 1) (10 μg/mL)-treated adultdermal fibroblasts. Interestingly, fibroblasts stimulated with EBPL-2demonstrated higher net deposition of tropoelastin than theircounterparts cultured with VGVAPG (SEQ ID NO.1).

Since EBPL-2 efficiently stimulated tropoelastin production, we nexttested whether this monomer precursor can be efficiently incorporatedinto the cross-linked insoluble elastin, a major component of elasticfibers. Results of metabolic labeling of cultured dermal fibroblastswith [³H]-valine, followed by assay of NaOH-insoluble elastin(normalized per DNA content) indicated that fibroblasts maintained for 7days in the presence of EBPL-2 or its acetylated version produce moreelastin (FIG. 7). Acetylating the N-terminus of EBPL-2 appeared toenhance its biological activity, although not statistically differentthan non-acetylated EBPL-2. On the other hand, amidating the C-terminusof EBPL-2 abolished its biological activity, indicating that theC-terminal glycine is imperative to the overall secondary structure ofEBPL-2 that permits binding to the EBP and induction of elastogenesis.Additional quantification of the newly deposited insoluble elastin(expression of the obtained results per culture dish) demonstrated aneven more spectacular net increase in metabolically labeled insolubleelastin (FIG. 8).

Results of this calculation, showing a more profound effect of EBPL-2 onnet deposition of insoluble elastin per culture, led to the hypothesisthat EBPL-2, similarly to VGVAPG (SEQ ID NO. 1), also stimulatescellular proliferation. This assumption was later confirmed by a directquantification of cellular proliferation showing higher than controlincorporation of [³H]-thymidine by dermal fibroblasts treated withEBPL-2 and VGVAPG (SEQ ID NO. 1).

Example 6

In a separate series of experiments we also tested whether proteinextracts of rice bran would reveal active GXXPG-like sequences capableof inducing elastogenesis in dermal fibroblast cultures. First, wedemonstrated by Western blotting that insoluble and soluble fractions ofrice bran contained GXXPG (SEQ ID NO. 2) epitopes that wereimmuno-reactive with anti-tropoelastin and anti-VGVAPG antibodies.Furthermore we found that a chemical digest of rice bran, which wasenriched in elastin-like NaOH-insoluble protein, demonstrated similarresults. Final experiments revealed that all rice bran-derivedpreparations enhanced deposition of tropoelastin by cultured adultdermal fibroblasts (FIG. 9).

Example 7

The following procedures, although not intended to limit the scope ofthe invention, were used to prepare chemically digested rice branextracts:

Protocol A

-   50 grams of soluble Rice Bran—(Nutri Rice CDC Group Inc)-   Dissolve in 160 ml 80% Ethanol+40 ml Acetone-   Incubation 30 Min in 37° C., Discard supernatant, and save the    pellet-   Dissolve the pellet in 100 ml of 1 M NaOH diluted in distilled    water,-   Boil for 60 min.-   Centrifuge at 4000 rpm for 10 min, discard supernatant-   Wash the pellet with distilled water 4 times-   Dissolve the pellet in 100 ml of 0.25M Oxalic acid-   Boil for 60 min-   Centrifuge at 4000 rpm for 10 min-   Collect supernatant 1-   Dissolve the pellet in 100 ml of 0.25M Oxalic acid-   Boil for 60 min-   Centrifuge at 4000 rpm for 10 min-   Collect supernatant 2-   Dissolve the remaining pellet in 50 ml of 0.25M Oxalic acid-   Boil for 60 min-   Centrifuge at 4000 rpm for 10 min-   Collect supernatant 3-   Combine collected supernatants 1, 2, 3-   Dialyze against 5 changes of distilled water-   Lyophilize-   Final powder: 230 mg, concentration of pure protein: 0.342 ug/mg

Protocol B

-   50 grams of Insoluble Rice Bran—(Nutri Rice CDC Group Inc.)-   Dissolve in 160 ml 80% Ethanol+40 ml Acetone-   Incubate 30 Min in 37° C., Discard supernatant, and save the pellet-   Dissolve the pellet in 400 ml of 1 M NaOH diluted in distilled    water,-   Boil for 60 min.-   Add 100 ml of 1 M NaOH diluted in distilled water-   Boil for 60 min-   Centrifuge at 4000 rpm for 10 min, discard supernatant-   Wash the pellet with distilled water 4 times-   Dissolve the pellet in 300 ml of 0.25M Oxalic acid-   Boil for 60 min-   Centrifuge at 4000 rpm for 10 min-   Collect supernatant 1-   Dissolve the pellet in 200 ml of 0.25M Oxalic acid-   Boil for 60 min-   Centrifuge at 4000 rpm for 10 min-   Collect supernatant 2-   Dissolve the remaining pellet in 150 ml of 0.25M Oxalic acid-   Boil for 60 min-   Centrifuge at 4000 rpm for 10 min-   Collect supernatant 3-   Combine collected supernatants 1, 2, 3-   Dialyze against 5 changes of distilled water-   Lyophilize (96 hr)-   Final powder: 201 mg, concentration of pure protein 8.89 ug/mg

Protocol C

-   100 mg Extracted soluble Rice Bran pellet-   Dissolve in 5 ml Amylase (1000 U/ml)-   Incubate 72 hr at 37° C. and shake-   Centrifuge at 1500 rpm for 10 min-   Collect supernatant-   Dialyze against 3 changes of distilled water-   Lyophilize-   Dissolve final powder in 200 ul distilled water, concentration of    pure protein 1.42 ug/ul

Protocol D

-   100 mg Extracted Insoluble Rice Bran pellet-   Dissolve in 10 ml Amylase (1000 U/ml)-   Incubate 72 hr at 37° C. and shake-   Centrifuge at 1500 rpm for 10 min-   Collect supernatant-   Dialyze against 3 changes of distilled water-   Lyophilize-   Dissolve final powder in 200 ul distilled water, concentration of    pure protein 0.16 ug/ul

Protocol E

-   10 grams of Soluble Rice Bran,—(Nutri Rice CDC Group Inc)-   10 grams of Insoluble Rice Bran,—(Nutri Rice CDC Group Inc-   Dissolve in 40 ml Amylase (1000 U/ml)-   Incubate 5 days at 37° C. and shake-   Dialyze against 3 changes of distilled water-   Add 40 ml 80% Ethanol+10 ml Acetone-   Incubate 30 Min at 37° C.,-   Centrifuge at 4000 rpm for 10 min and save the pellet-   Dissolve the pellet in 40 ml of 1 M NaOH diluted in distilled water,-   Boil for 60 min.-   Centrifuge at 4000 rpm for 10 min, discard supernatant-   Wash the pellet with distilled water 4 times-   Dissolve the pellet in 40 ml of 0.25M Oxalic acid-   Boil for 60 min-   Centrifuge at 4000 rpm for 10 min-   Collect supernatant 1-   Dissolve the pellet in 40 ml of 0.25M Oxalic acid-   Boil for 60 min-   Centrifuge at 4000 rpm for 10 min-   Collect supernatant 2-   Dissolve the remaining pellet in 20 ml of 0.25M Oxalic acid-   Boil for 60 min-   Centrifuge at 4000 rpm for 10 min-   Collect supernatant 3-   Combine collected supernatants 1, 2, 3-   Dialyze against 5 changes of distilled water-   Lyophilize (96 hr)-   Take 5 mg of Final powder and dissolve in 100 ul distilled water-   Concentration of protein:    -   Soluble rice: 3.44 ug/ul    -   Insoluble rice: 0.217 ug/ul

Protocol F

-   3 grams of Soluble Rice Bran,—(Nutri Rice CDC Group Inc)-   3 grams of Insoluble Rice Bran,—(Nutri Rice CDC Group Inc)-   Make 70% formic acid and bubble nitrogen through it to remove the    oxygen (CNBr does not work in the presence of oxygen)-   In the fume hood, using a disposable tube weight 2 gm of Cyanogen    Bromide (CNBr) to make a 40 ml solution when dissolve in the formic    acid (50 mg/ml)-   Add 20 ml of solution to 3 gm soluble rice bran and 3 gm insoluble    rice bran respectively-   Close lid and leave in the fume hood to digest overnight-   Next day, use boiling water to wash digests 5 times in the fume hood-   Centrifuge at 3300 rpm for 10 min in the fume-   Save pellet-   Half of pellet should be lyophilized-   Half of pellet should be dissolve in 20 ml 1 M NaOH and boiled for    60 min-   For Soluble rice pellet: Add 30 mL distilled water and dialyze    against 3 changes of distilled water-   lyophilize-   For Insoluble Rice pellet:-   Wash with distilled water 3 times-   Dialyze against 3 changes of distilled water-   Lyophilize-   Take 2 mg of each final powder, dissolve in 100 ul distilled water,-   Concentration of protein:-   1, CNBr digested soluble rice: 3.7 ug/ul-   2, CNBr digested insoluble rice: out of range-   3, CNBr+NaOH Soluble rice: out of range-   4, CNBr+NaOH Insoluble rice: 7.03 ug/ul

Protocol G

-   Starting material: 1 gram of Soluble Rice Bran (Nutri Select    Soluble—Nutri Rice CDG Group Inc.)-   Dissolve in 40 ml of 1 M NaOH diluted in 80% Ethanol-   Centrifuge at 4000 rpm for 10 min. and save the pellet-   Dissolve pellet in 20 ml of distilled water-   Dialyze overnight against distilled water (MWCO 2000)-   Lyophylize (48 hours)-   Dissolve the lyophilized powder in 5 ml of 0.25M Oxalic acid-   Boil for 1 hour-   Centrifuge at 4000 rpm for 10 min.-   Collect supernatant 1-   Dissolve the pellet in 5 ml of 0.25M Oxalic acid-   Boil for 1 hour-   Centrifuge at 4000 rpm for 10 min-   Collect supernatant 2-   Dissolve the remaining pellet in 5 ml of 0.25M Oxalic acid-   Boiling for 1 hour-   Centrifuge at 4000 rpm for 10 min-   Collect supernatant 3-   Combine collected supernatants 1, 2, 3-   Dialyze against 5 changes of water-   Lyophylize (48 hours)

Both Amylase-digested soluble and insoluble rice bran preparationscontain proteins that react with antibody raised to human tropoelastin(FIG. 10). Additionally, the soluble bran preparation also contains apeptide with the unique AKAAAKAAAKA (SEQ ID NO. 15) domain detected intropoelastin.

Western blotting with anti-tropoelastin antibody demonstrated thatsamples of CNBr digests of rice bran preparations contain protein(s)with similar immunoreactivity as human 70-kDa tropoelastin and itsdegradation products (FIG. 11). 1: Cyanogen bromide digest of solublerice bran. 2: Cyanogen bromide digest of insoluble rice bran. 3: Extractfrom culture of elastin-producing human fibroblasts.

Example 8

FIG. 12 shows representative micrographs of 10-day old organ cultures ofhuman skin explants (44 year woman) stained with Movat pentachromestain. Cultures treated with EBPL-2 contained more elastic fibers (blackstain) produced by cells migrating out of the basal layer of theepidermis (upper panel) and by myofibroblasts localized in the deeperdermal zone (lower panel).

Example 9

EBPL-2 stimulates dilatation of existing capillaries in organ culturesof human skin biopsies. FIG. 13 shows multiple micrographs demonstratingthat treatment with EBPL-2 induce dilatation of capillaries whichpericytes produce elastic fibers.

EBPL-peptides stimulate elastogenesis in human heart stromal cells. Themicrographs of FIG. 14 demonstrate that synthetic EBPL peptidessignificantly increase production of elastic fibers by stromal cellsisolated from human heart.

Although the present invention has been described in considerable detailwith reference to certain preferred embodiments thereof, other versionsare possible. Therefore the spirit and scope of the appended claimsshould not be limited to the description and the preferred versionscontain within this specification.

What is claimed is:
 1. A synthetic sextapeptide comprising a sequence ofIGVAPG (SEQ ID NO: 13).
 2. The synthetic sextapeptide of claim 1,wherein the sextapeptide provides an appearance of increasedelastogenesis in a tissue.
 3. The synthetic sextapeptide of claim 1,wherein the sextapeptide stimulates elastogenesis.
 4. A method ofstimulating elastogenesis comprising administering a therapeuticallyeffective amount of a composition comprising a synthetic peptide havinga sequence selected from VGVAPGAAAAAVGVAPG (SEQ ID NO: 10), IGVAPG (SEQID NO: 13), and IGVAPGAAAAAIGVAPG (SEQ ID NO: 14).
 5. The method ofclaim 4, wherein the composition provides an appearance of increasedelastogenesis in a tissue.
 6. The method of claim 4, wherein thecomposition stimulates proliferation and migration of dermal fibroblastsinto an area of skin.
 7. The method of claim 4, wherein the compositionprovides an enhanced deposition of elastin in a tissue.
 8. The method ofclaim 4, wherein the composition improves the appearance of skin.
 9. Themethod of claim 4, wherein the administering includes at least one oftopical application and application via a transdermal patch.
 10. Amethod of stimulating elastogenesis comprising administering atherapeutically effective amount of a composition comprising a syntheticsextapeptide having a sequence of IGVAPG (SEQ ID NO: 13).